(1) Field of the Invention
The present invention relates to an apparatus comprising one or more piezoelectric mass sensors for use in diagnostic and analytic processes, in particular for immunochemical detection of diagnostically relevant analytes. Each piezoelectric mass sensor comprises a piezoelectric crystal with a receptor surface which has immobilized thereon a lawn of recombinant antibodies comprising single VH chain or single-chain Fv (scFv) polypeptides specific for a particular antigen. Binding of antigen to the recombinant antibodies results in a change in mass on the receptor surface which is detected as a change in resonant frequency. In a preferred embodiment, the receptor layer is a precious metal such as gold which facilitates self-assembly of the recombinant antibodies into a lawn on the receptor surface via a cysteine residue at the carboxy terminus of the attachment polypeptide.
(2) Description of Related Art
In 1880, Pierre and Jacques Curie observed that pressure exerted on a small piece of quartz causes an electrical potential difference between the deformed surfaces, and found that application of a voltage to a quartz crystal caused physical distortion (Lu C, Czanderna A W. edt, Methods and Phenomena, Volume 7, Elsevier, New York, 1984, 198-280). They named their discovery the piezoelectric or “pressure electric” effect.
The piezoelectric property characteristic of quartz is only possible in ionic crystalline solids lacking a center of inversion. Of the 32 three-dimensional point groups, only twenty can possibly exhibit the piezoelectric effect; however in some it may be too small to detect. It was found that α-quartz exhibited the piezoelectric effect and, because of its mechanical and thermal stability, is used to construct highly stable oscillator circuits with pg/cm2 mass sensitivity.
Depending on the cut-angle, a large number of different resonator types can be obtained from α-quartz. The mode of vibration that is most sensitive to the addition or removal of mass from a quartz crystal is the thickness shear mode. To make a quartz crystal oscillate in the thickness shear mode, the crystal must be cut at a specific angle with respect to the principal optical axes of the quartz. AT-cut quartz crystals cut at an angle of 35.25° to the z-axis exhibit a high frequency stability (Δf/f=10−8) and almost zero temperature coefficient between 0-50°, and are most frequently used for mass-sensing devices.
Interfacial mass changes can be related to changes in the QCM oscillation frequency by applying Sauerbrey's equation (G. Sauerbrey., Z. Phys. 1959; 155:206-222), Δf=−2Δmnf02/(A(μqρq)1/2), where n is the overtone number, μq is the shear modulus of the quartz (2.947×1011 g/(cm sec2), and ρq is the density of the quartz (2.648 g/cm3), and which assumes the foreign mass is strongly coupled to the resonator.
Mass detection by using Sauerbrey's equation is usually straightforward if the device is operating in the gas or the vacuum phase, the added mass binds tightly to the surface, and the films of added mass are stiff and thin, such as in electroplating. Because of their small size, high sensitivity, and stability, piezoelectric crystals have been used as microbalances in the determination of thin-layer thickness and in general gas-sorption studies (Grate J W, Martin S J, White R M., Anal Chem 1993; 65:940A-948A and Lu C, Czanderna A W. edt, Methods and Phenomena, Volume 7, Elsevier, New York, 1984, 198-280).
The use of QCM in analytical applications was delayed due to lack of suitable oscillator circuits that enable the shear-wave resonator to be operated in fluids. In 1982, Nomura and Okuhara were the first to report on a circuitry capable of oscillating in liquid (Nomura T, Okuhara M. Anal Chim Acta. 1982; 142;281-284). This gave the starting point for development of a new class of analytical tools. The incorporation of various chemically sensitive layers has resulted in the explosive growth of piezoelectric sensors (Janshoff A, Galla H, Steinem C. Angew Chem. Int. Ed. 2000; 39:4004-4032). The major advantages of piezoelectric mass sensors are simplicity of construction and operation, weight, cost, availability, and low power requirements. Unlike electrochemical sensors, the measurement is conducted in a monopolar mode, i.e., only a single physical probe is necessary. Mass sensors have high sensitivity and can be used for a broad range of compounds.
In recent years, methods based on the use of piezoelectric crystal devices have been developed for immunoassay applications (Guilbault G G, Hock B, Schmid R., Biosensors Bioelectronics. 1990; 5:13-26; Schmitt N, Tessier L, Watier H, Patat F, Sensors and Actuators B. 1997; 43:217-223 and Su X, Chew F T, Li S F Y., E. Anal Biochem. 1999; 273:66-72). However, researchers are still skeptical about the potential of piezoelectric mass sensing devices as biosensors. Rodahl et. al (Rodahl M, Hook F, Fredriksson C, Keller C A, Krozer A, Brzezinski P, Voinova M, Kasemo B. Faraday Discussions. 1997; 107:229-246) studied protein adsorption, lipid vesicle adsorption, and cell adhesion on QCM electrode. Their results demonstrated that even thin biofilms dissipate a significant amount of energy owing to QCM oscillation. They attribute the measured increase in energy dissipation to (1) a viscoelastic porous structure that is strained during oscillation, (2) trapped liquid that moves between or in and out of pores due to the deformation of the film, (3) the load from the bulk liquid which increases the strain of film.
The physics of biofilms in liquid is complex, which makes it difficult to obtain a generally explicit relationship between the added mass and the change in the frequency output. QCM may give a direct response signal that characterizes the binding event between a sensitive layer, immobilized on the surface of transducer, and the analytes to be detected. However, the mass estimated with the QCM response through the Sauerbrey equation and the mass measured can be quite different. Several papers demonstrated that the deposited mass is generally overestimated (Babacan S, Pivarnik P, Letcher S, Rand A G. Biosensors & Bioelectronics. 2000; 15:615-621 and Bizet K, Gabrielli C, Perrot H, Therasse J, Biosenors & Bioelectronics. 1998; 13:259-269). Another limitation of QCM biosensors arises from the large size of biomolecules such as immunoglobulins. Consequently, low densities of the binding molecule are usually immobilized on the surface. A signal will only be obtained if the interaction results in a net change of mass of the selective protein layer attached to the crystal. If the interaction is a displacement of one species with another, i.e., the exchange or catalytic reaction, the sensor surface is only a temporary host to the interacting species and the net changes of mass can be very small. For small biomolecules, such as some antigens, it is quite difficult to obtain an observable signal due to the small amount of sensitized molecule immobilized and limited sensitivity of commonly used 5 MHz and 10 MHz quartz crystal.
The above concerns did not stop researchers' enthusiasm for piezoelectric sensors. In the past decade, numerous studies have shown that adsorption of biomolecules on functionalized surface is one of the paramount applications of piezoelectric transducers. Examples include the study of the interaction of DNA and RNA with complementary strands (Okahata Y, Kawase M, Niikura K, Ohtake F, Furusawa H, Ebara Y. Anal Chem. 1998; 70:1288-1296), specific recognition of protein ligands by immobilized receptors, and the detection of virus capsids, bacteria, and mammalian cells (Fredriksson C, Kihlman S, Rodahl M, Kasemo B. Langmuir. 1998; 14:248-251). However, whether QCM will assert itself against established label-free sensors such as surface plasma resonance spectroscopy and interferometry rests on development of a functionalized film on quartz which is thin, rigid, and contains a high density of the sensing molecules.
The most sensitive analytic apparatus yet developed is the piezoelectric immunosensor which has the potential capability of detecting antigens in the picogram range. In addition, the piezoelectric immunosensor is believed to have the potential to detect antigens in or from the gas phase as well as in the liquid phase. The state of the art related to piezoelectric immunosensors are exemplified by the following U.S. patents.
U.S. Pat. No. 4,236,893 to Rice discloses an apparatus and method for performing immunoassays for detecting particular classes of antibodies in a liquid sample using a piezoelectric oscillator. The oscillator comprises a quartz crystal coated with an antigen recognized only by a particular class of antibody. The coated oscillator is incubated in the liquid sample for a time sufficient for the antibody to bind the antigen. Afterwards, the oscillator is removed from the sample, washed and dried, and the resonant frequency measured. A change in resonant frequency indicates the sample contained the particular class of antibody specific for the antigen.
U.S. Pat. No. 4,242,096 to Oliveira et al. discloses an indirect immunoassay for detecting an antigen in a liquid sample using a piezoelectric oscillator. The oscillator comprises a quartz crystal coated with an antigen to be detected. The coated oscillator is incubated in the liquid sample to which a predetermined amount of antibody specific for the antigen has been added for a time sufficient for the antibody to bind either the antigen in the sample or the antigen on the quartz crystal. Afterwards, the oscillator is removed from the sample and the resonant frequency measured. The amount of change in resonant frequency indirectly indicates the amount of antigen present in the liquid sample.
U.S. Pat. No. 4,246,344 to Silver III discloses a method for detecting adherent cells using a piezoelectric oscillator. The resonant frequency of a piezoelectric oscillator is determined and then incubated in a liquid sample for a time sufficient for adherent cells to adhere the oscillator. Afterwards, the oscillator is removed from the sample, washed and dried, and the resonant frequency determined. A change in resonant frequency indicates that the sample contains adherent cells.
U.S. Pat. No. 4,314,821 to Rice discloses an apparatus and method for performing immunoassays for detecting an antibody in a liquid sample using a piezoelectric oscillator. The oscillator comprises a quartz crystal coated with an antigen recognized by the antibody. The coated oscillator is incubated in the liquid sample for a time sufficient for the antibody to bind the antigen. Afterwards, the oscillator is removed from the sample, washed and dried, and the resonant frequency measured. A change in resonant frequency indicates the sample contained an antibody specific for the antigen.
U.S. Pat. No. 4,735,906 to Bastiaans discloses an apparatus and method for performing immunoassays for detecting an analyte using a piezoelectric sensor. The sensor comprises a piezoelectric crystal coated with a monomer layer of a silane derivative to which a member of a specific binding pair for the analyte is chemically bonded. When the sensor is incubated with a liquid sample containing the analyte, the analyte binds to the specific binding pair which then causes a change in the resonant frequency of the sensor.
U.S. Pat. No. 5,314,830 to Anderson et al. discloses a method for immobilizing an antibody on a surface such as the surface of the crystal comprising a piezoelectric oscillator. An antibody modified with a hydrophobic moiety attached to the antibody by a spacer comprising a water soluble polymer is directly absorbed to the surface of the surface.
U.S. Pat. No. 5,932,953 to Drees et al. discloses a method and system for detecting a material bound on a surface of a piezoelectric resonator. The method uses a sensing resonator that measures a change in insertion phase shift of the resonator caused by binding of the material being detected on the surface of the resonator instead of measuring the change in the oscillation frequency of the sensing resonator caused by the binding of the material being detected on the surface of the resonator.
U.S. Pat. No. 6,087,187 to Wiegland et al. discloses a method for using a piezoelectric sensor for the immunochemical detection of an analyte in a liquid sample. The piezoelectric sensor comprises a precious metal coating on the surface to which a specific binding partner is bound. Preferably, the specific binding partner is an antibody, antibody fragment, a lectin, or an antigen. The sensor is incubated with the sample for a time sufficient for the specific binding partner to bind the analyte which causes a shift in the resonant frequency of the sensor. Afterwards, the specific binding partner and bound analyte are removed from the surface.
Published U.S. Patent Application Nos. 20030077222, 20030073133, 20030072710, 20030068273, 20030053950, and 20030049204, all to Leyland-Jones, discloses immunosensors which in particular embodiments have antibodies, Fab fragments, or scFv polypeptides immobilized on the surface thereof.
Currently available biosensors as exemplified by the above U.S. patents provide accurate detection but have significant disadvantages in terms of cost, time needed for detection, lack of portability, ability to function in a “dirty” environment, and the need for highly trained technicians to operate the systems. Piezoimmunosensor (PZ) technology, which places antibodies on a quartz crystal microbalance (QCM) to detect minute changes in mass as the antibodies bind with antigens may address these drawbacks. However, use of piezoelectric technology in biosensors is problematic due to the complex nature of whole antibodies. For example, the large size and branching arms of whole antibodies increase their susceptibility to proteases and non-specific binding and trapping of antigen, which reduces sensitivity and accuracy. In addition, polyclonal antibodies are difficult to use because of their heterogeneous nature and monoclonal antibodies, while affording homogeneous binding characteristics, are labor intensive and expensive to produce.
Therefore, there is a need for a biosensor which provides sensitive and accurate detection but which does not have the drawbacks inherent in biosensors which use whole antibodies.